EP0075925B1 - Procédé de préparation d'un régulateur de l'appétit agissant spécifiquement sur le centre de la nutrition, et le composé obtenu par le procédé - Google Patents

Procédé de préparation d'un régulateur de l'appétit agissant spécifiquement sur le centre de la nutrition, et le composé obtenu par le procédé Download PDF

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Publication number
EP0075925B1
EP0075925B1 EP82108965A EP82108965A EP0075925B1 EP 0075925 B1 EP0075925 B1 EP 0075925B1 EP 82108965 A EP82108965 A EP 82108965A EP 82108965 A EP82108965 A EP 82108965A EP 0075925 B1 EP0075925 B1 EP 0075925B1
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Prior art keywords
gel
solution
concentration
weight
active ingredient
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Expired - Lifetime
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EP82108965A
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German (de)
English (en)
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EP0075925A2 (fr
EP0075925A3 (en
Inventor
József Dr. Knoll
János Dr. Dipl. Chem. Nagy
Huba Dr. Dipl. Chem. Kalász
Berta Dr. Knoll
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Richter Gedeon Vegyeszeti Gyar Nyrt
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Richter Gedeon Vegyeszeti Gyar RT
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Priority to AT82108965T priority Critical patent/ATE59556T1/de
Publication of EP0075925A2 publication Critical patent/EP0075925A2/fr
Publication of EP0075925A3 publication Critical patent/EP0075925A3/de
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S530/00—Chemistry: natural resins or derivatives; peptides or proteins; lignins or reaction products thereof
    • Y10S530/827—Proteins from mammals or birds
    • Y10S530/829—Blood
    • Y10S530/83—Plasma; serum

Definitions

  • the invention relates to a method for producing an appetite-regulating active substance from human and / or animal blood serum which acts specifically on the nutritional center, and the active substance obtained for use as an appetite regulator.
  • Hungarian patent specification 178 703 already discloses an appetite-regulating active substance which acts specifically on the nutritional center and is isolated from human and / or animal blood.
  • the eluate is collected in fractions, the biologically active fractions are evaporated, the residue is dissolved in the required amount of water, the solution is chromatographed again on a gel of the type mentioned and fractionally eluted with water, and the fractions exhibiting biological activity are evaporated.
  • a fraction which appeared to be separable by hydrolysis into amino acid and sugar constituents (about 60% by weight of amino acids and 10% by weight of sugar) as a glycoproteid could be separated off, which fraction acts as an appetite-regulating active substance which acts specifically on the nutritional center the central nervous system was neither stimulating nor depressing.
  • the invention is therefore based on the object of a method for producing an appetite-regulating active substance specifically acting on the nutrition center from human and / or animal blood serum, by means of which a purer active substance with a better appetite-regulating effect can be obtained, and the active substance obtained for use as an appetite regulator to accomplish.
  • the fraction which can be prepared by the known process mentioned can be further broken down by means of a new process and a new product which is 3 to 4 times as effective as the fraction separated by the known process can be obtained .
  • the chemical composition of this product differs significantly from that of the known fraction.
  • the product is to be regarded as a physically homogeneous, chemically pure substance with a defined composition.
  • the protein or peptide content of the active ingredient fraction separated by the known process mentioned is present to a significant extent in chemically unbound or only loosely bound form as an inert component with respect to the satietin effect and can be separated from the actual active ingredient by means of appropriate protein precipitation procedures.
  • the remaining low molecular weight, peptide-like and other accompanying substances can be completely removed by further purification stages, namely by means of electrophoresis and by means of affinity chromatography. In this way, the pure chemically uniform active ingredient satietin can be produced.
  • the invention therefore relates to a process for the production of an appetite-regulating active substance which acts specifically on the nutritional center from human and / or animal blood serum by filtering its membrane through a membrane filter which is permeable to a molecular weight of 50,000 and evaporating the filtrate and gel-chromatographing a solution of the material obtained a gel with an exclusion volume M below 50,000 daltons and fractional elution with an aqueous solution, re-gel chromatography of a solution of the concentrated biologically active fractions on a gel with an exclusion volume M below 50,000 daltons and fractional elution with water and concentration of the biologically active fractions, which characterized in that the filtrate obtained in membrane filtration is only partially evaporated (concentrated) and the insoluble part is removed from the concentrate obtained, then contributes to the liquid phase 0 to 10 ° C., an aqueous trichloroacetic acid solution is added until a trichloroacetic acid concentration of 5 to 25% by weight is reached and the precipit
  • the removal of the insoluble part of the concentrate obtained after partially evaporating the filtrate from the membrane filtration is carried out by centrifugation.
  • a sodium chloride solution a concentration of 0.1 to 1.0% by weight, preferably 0.9% by weight, can be used for elution.
  • the first cleaning stage of the method according to the invention membrane filtration (ultrafiltration), can be carried out, for example, with Amicon® UM-10 or Sartorius® membranes, advantageously under a pressure of 3 atm with constant stirring.
  • the filtrate obtained is advantageously partially evaporated by vacuum evaporation and thus concentrated, and the insoluble part is preferably removed by centrifugation.
  • the cloudy, but precipitation-free solution obtained is cooled to 0 to 10 ° C. and then with an aqueous trichloroacetic acid solution to a concentration of 5 to 25% by weight, preferably 10 to 12% by weight, in particular approximately 10% by weight, added.
  • the solution in which a precipitate separates is left at 0 to 5 ° C for at least 1 hour, but preferably overnight, and then the precipitated protein is separated by ultracentrifugation at the same temperature, a clear, vivid yellow solution being obtained.
  • the treatment with the trichloroacetic acid solution removes all high-molecular serum proteins, including the albumin.
  • the satietin which has a high carbohydrate content, does not precipitate, but remains in solution, although it still contains a small amount of protein. It follows that this amount of protein must already be in chemically bound form, namely on the carbohydrate part of the satietin.
  • Columns filled with Sephadex® G-15 or Sephadex o G-25 are preferred.
  • An aqueous 0.1 M ammonium acetate buffer solution with a pH of 6.6 is preferably used for elution.
  • the fractions with satietin activity could be separated most effectively with this buffer solution and a practically salt-free product is obtained due to the volatility of the buffer.
  • the active fraction appears at the exclusion volume of the gel column (V., at which K d , the distribution coefficient of the investigated substance, is 0).
  • V. the exclusion volume of the gel column
  • K d the distribution coefficient of the investigated substance
  • the cleaning effectiveness of this cleaning step corresponds to entire orders of magnitude.
  • the trichloroacetic acid used to precipitate the proteins is retained as a low-molecular compound in the gel column.
  • salt-free and acid-free active ingredient is obtained.
  • Bio-Gel P-2 0 manufactured by Bio-Rad Laboratories, Richmond, California, USA] is preferably used as gel and distilled water is used as water for the eluent.
  • the salts and low molecular fragments still contained in the product for example fragments of peptides, are removed.
  • the fractions with satietin activity are not absorbed by the gel and appear in the eluate volume corresponding to the column volume.
  • the active ingredient is obtained in the form of a pale yellow powder which is easy to handle.
  • 8 to 10 mg of lyophilized product can be obtained from 1 I of human blood serum (corresponds to I of ultrafiltrate).
  • the raw satietin obtained in this way can be regarded as a standard raw product, but it is also sufficiently pure in this form for practical purposes, that is to say to regulate the appetite. Its satietin activity is 25 to 50 SE / mg.
  • 1 satietin unit is the amount of active ingredient which, when administered to 96 hours of starving, 200 to 240 g female CFY rats intracerebroventricularly reduced their consumption of standard food discs on the first day of feeding from the average 24.04 ⁇ 0.76 g to 10 g.
  • raw satietin can be obtained not only from human blood, but also from animal blood serum, such as blood sera from cattle, horses, rabbits or rats.
  • animal blood serum such as blood sera from cattle, horses, rabbits or rats.
  • the yield and potency of the product obtained may differ (for example, a little more product [10 to 13 mg / l] is obtained from the serum of bovine blood than from the serum of human blood using the same procedure, but it has the same effectiveness as that of the product obtained from human blood), the specific selectively responsive nature of the nutritional mechanism of the effect is the same for products of different origins.
  • the molecular weight, determined by means of gel electrophoresis with sodium n-dodecyl sulfate (SDS) on polyacrylamide and by means of gradient electrophoresis on polyacrylamide gel, of the crude satietins obtained having an activity of 25 to 50 SE / mg is 50,000 to 70,000. It is salt-free , contains practically no albumin, has a low protein content (5 to 25% by weight) and a high carbohydrate content (60 to 90% by weight) and is a pale yellow powder in the lyophilized state.
  • the product contains the 4 main sugar components fucose, mannose, galactose and glucose in the carbohydrate part.
  • glucosamine is detectable in the hydrolyzed product.
  • the gel electrophoresis carried out with sodium n-dodecyl sulfate on polyacrylamide and the analytical isoelectric focusing show 4 to 6 strips (constituents or subunits) which can be seen by protein coloring.
  • the product was further subjected to medium-voltage electrophoresis in buffer solutions with a pH of 6.2 or 1.6 and was still found to be a mixture.
  • the main biologically active component remains at the point of application or moves very little in the direction of the cathode.
  • the active substance can be detected with ninhydrin or iodic acid, which also speaks for the glucoproteid nature of the substance.
  • the crude satietin obtained in the manner described can be further purified electrophoretically, advantageously on a laboratory scale by paper electrophoresis.
  • the active ingredient obtained in this way is already very pure.
  • the substance was shown to be uniform.
  • the activity of the substance essentially showing the character of glucoproteid is approximately 100 S.E./mg.
  • the pure substance can be obtained if the active ingredient which has been separated off and electrophoretically purified in the manner described is used in a further separation step in the recently developed affinity chromatography (see, for example, DM Swallow, L. Evans, DA Hopkinson: Nature g269 [1977], 261 to 262, in which scripture also all substances mentioned in connection with affinity chromatography appear in this text). This can expediently be carried out as follows.
  • a substance which can selectively bind a group present in the substance to be separated but not contained in the accompanying substances must be used as the adsorbent.
  • An adsorbent which specifically binds the glucopyranose groups of the glucoproteides can be used for the satietin which has been found to be a glucoproteide.
  • the adsorbent gel known as "Con-A-Sepharose" has proven to be the most advantageous.
  • This gel in which is coupled to Sepharose 4B Concavalin A activated with cyanogen bromide, binds under certain conditions the molecules having an aD-mannopyranosyl or aD-glucopyranosyl group, while the accompanying substances, such as other proteins and peptides, which do not contain these groups, bind to the gel not be bound. These non-bound substances appear in the first eluate approximately in the! the amount corresponding to the column volume.
  • the glucoproteid-like substances bound to the gel can be separated further depending on the type of binding, that is to say their chemical properties, especially if the substances bound to the gel are eluted with a gradient.
  • the substance which has already been largely purified by fractionation in the manner specified is dissolved in a starting buffer solution of neutral reaction.
  • An aqueous 0.02 m 2- (amino) -2- (hydroxymethyl) propane-1,3-diol hydrochloride solution [tris hydrochloride solution], which contains sodium chloride in a concentration of 0.5 to 1.0 mol / 1 and advantageously has a small amount (about 1 mmol) of Ca ++ - and Mn ++ - containing ions used.
  • the relatively high salt concentration in the starting buffer solution is necessary to prevent non-specific protein binding between the adsorbent and the accompanying substances with protein character.
  • the substance dissolved in the starting buffer solution is applied to the column filled with the adsorbent, preferably Con-A-Sepharose.
  • the column is previously with the The starting buffer solution was brought into equilibrium, for example in such a way that at least 10 times the column volume of buffer solution was passed through.
  • elution is carried out using a concentration gradient, namely a buffer solution containing a-methylmannoside in increasing concentration. It is advantageous to use an elution solution of the same composition as that of the starting buffer solution, but as already mentioned, containing a-methylmannoside.
  • An a-methylmannoside solution with a concentration of 0.5 mol / l is preferably continuously added dropwise to the buffer solution and the mixture is stirred continuously. A linear gradient is obtained in this way. Only the content of a-methylmannoside changes during the elution, whereas the ion concentration and the pH of the solution are not changed during the elution.
  • the process is followed at a wavelength of 254 nm with a registration photometer, the elution diagram shown in the drawing, according to which the mixture of substances representing tip 1 passes through the column and appears in the initial fraction, while the glucoprotein bound to the column appears with a much larger retention value in the form of the almost symmetrical tip 2 is eluted to the extent that the a-methylmannoside concentration of the gradient increases.
  • the appropriate fractions are pooled and concentrated; Since the substance obtained in this way still contains the salts, a-methylmannoside and possibly also low-molecular fragments which have been split off from the high-molecular-weight active substance, a final gel chromatography is necessary to separate the active substance in order to separate it.
  • a gel with an exclusion volume below M 3,000 Dalton, advantageously the "Bio-Gel P-2" gel, is used. It is eluted with ion-free water. The salts and other low-molecular impurities still contained in the product enter the interior of the gel due to their small molecular size and are retained there. The pure salt-free satietin is washed from the surface of the gel and appears in the first eluate corresponding to the column volume. The active ingredient-containing fractions obtained with pure ion-free water are combined and lyophilized; in this way the completely pure and uniform active ingredient satietin is obtained in the form of a white powder.
  • the pure satietin separated according to the invention has a molecular weight of 60,000 to 70,000, such as it was determined by gel electrophoresis with sodium n-dodecyl sulfate (SDS).
  • SDS sodium n-dodecyl sulfate
  • the analysis of the acidic hydrolyzate of the product showed the following values:
  • the analytical values refer to the lyophilized product. Depending on the circumstances of the lyophilization, the water content may differ.
  • the gradient electrophoresis on polyacrylamide gel and the isoelectric focusing it is a chemically homogeneous substance.
  • the invention also relates to the active ingredient obtainable by the process according to the invention. It can be used advantageously as an appetite suppressant. Compared to the known impure satietin product, it has the surprising great advantage that it is 3 to 4 times as effective as the latter.
  • the invention is illustrated by the following example.
  • the column was eluted with distilled water and the fractions between 130 and 180 cm 3 were combined and lyophilized. In this way, 25 to 30 mg of crude salt-free satietin was obtained in the form of a whitish-yellow powder.

Claims (7)

1. Procédé pour préparer une substance active agissant de façon spécifique sur le centre de la nutrition et réglant l'appétit, à partir d'un sérum sanguin humain et/ou animal au moyen d'une filtration à travers une membrane à l'aide d'un filtre à membrane perméable jusqu'à un poids moléculaire de 50 000, et d'une évaporation du filtrat ainsi qu'une chromatographie d'une solution de la substance obtenue, sur un gel possédant un volume d'exclusion M inférieur à 50 000 daltons, et d'une élution fractionnée avec une solution aqueuse, d'une nouvelle chromatographie sur gel d'une solution concentrée, active du point de vue biologique, sur un gel avec un volume d'exclusion M inférieur à 50 000 daltons et d'une élution fractionnée avec de l'eau, et d'une concentration des fractions actives du point de vue biologique, caractérisé en ce qu'on évapore seulement partiellement le filtrat obtenu lors de la filtration à l'aide de la membrane et qu'on élimine, du concentré obtenu, la partie insoluble, qu'on ajoute ensuite à la phase liquide, entre 0 et 10°C, une solution d'acide trichloracétique aqueux jusqu'à l'obtention d'une concentration de 5 à 25% en poids/volume de l'acide trichloracétique, et qu'on élimine la protéine qui a précipité, et qu'on exécute la chromatographie de la solution obtenue sur un gel possédant un volume d'exclusion inférieur à M = 4 000 daltons et qu'on réalise l'élution avec une solution de chlorure de sodium aqueuse intervenant pour 0,5 à 1,0% en poids ou avec une solution tampon aqueuse possédant un pH compris entre 6,0 à 7,0, que l'on concentre les fractions, actives du point de vue biologique, par liophylisation, qu'on exécute une nouvelle chromatographie du concentré obtenu sur un gel possédant un volume d'exclusion à M = 3 000 daltons et que l'on concentre les fractions éluées avec l'eau et actives du point de vue biologique, par lyophilisation et qu'on soumet le produit obtenu à une épuration ultérieure de manière à exécuter une électrophorèse réalisée avec un pH possédant une valeur comprise entre 6,0 et 6,5, et qu'on sépare le produit principal resté au point de départ et qu'on le soumet à une chromatographie par affinité, moyennant l'utilisation d'un agent adsorbant présentant une affinité spécifique vis-à-vis de groupes glucopyranose et/ou mannopyranose.
2. Procédé selon la revendication 1, caractérisé en ce qu'on utilise de la Con-A-Sépharose.
3. Procédé selon la revendication 1 ou 2, caractérisé en ce qu'on réalise par centrifugation l'élimination de la partie insoluble du concentrat obtenu après l'évaporation partielle du filtrat fourni par la filtration à travers la membrane.
4. Procédé selon l'une des revendications 1 à 3, caractérisé en ce qu'on réalise l'adjonction de la solution d'acide trichloracétique jusqu'à l'obtention d'une concentration comprise entre 10 et 12% en poids/volume.
5. Procédé selon la revendications 1 à 4, caractérisé en ce qu'on exécute par centrifugation l'élimination de la protéine qui précipite lors de l'adjonction de l'acide trichloracétique.
6. Procédé selon l'une des revendications 1 à 5, caractérisé en ce que pour l'élution, on utilise comme solution de chlorure de sodium, une telle solution possédant une concentration de 0,9% en poids.
EP82108965A 1981-09-28 1982-09-28 Procédé de préparation d'un régulateur de l'appétit agissant spécifiquement sur le centre de la nutrition, et le composé obtenu par le procédé Expired - Lifetime EP0075925B1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT82108965T ATE59556T1 (de) 1981-09-28 1982-09-28 Verfahren zur herstellung eines spezifisch auf das ernaehrungszentrum wirkenden appetitregelnden wirkstoffes sowie der nach diesem verfahren erhaeltliche wirkstoff.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
HU812783A HU183590B (en) 1981-09-28 1981-09-28 Process for the isolation of an active suastance influencing specifically the nutrition centre with a regulative effect on the appetite from human and/or animal blood serum
HU278381 1982-03-03

Publications (3)

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EP0075925A2 EP0075925A2 (fr) 1983-04-06
EP0075925A3 EP0075925A3 (en) 1986-01-02
EP0075925B1 true EP0075925B1 (fr) 1991-01-02

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EP82108965A Expired - Lifetime EP0075925B1 (fr) 1981-09-28 1982-09-28 Procédé de préparation d'un régulateur de l'appétit agissant spécifiquement sur le centre de la nutrition, et le composé obtenu par le procédé

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US (1) US4430264A (fr)
EP (1) EP0075925B1 (fr)
JP (1) JPS58135816A (fr)
AT (1) ATE59556T1 (fr)
AU (1) AU551250B2 (fr)
DE (1) DE3280277D1 (fr)
DK (1) DK429082A (fr)
HU (1) HU183590B (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT391807B (de) * 1982-05-21 1990-12-10 Solco Basel Ag Verfahren zur gewinnung zellatmungsfoerdernder wirkstoffe aus kaelberblut

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HU194916B (en) * 1983-07-29 1988-03-28 Richter Gedeon Vegyeszet Process for producing new type of active compound of selective inhibiting activity for intake of food
HUT45903A (en) * 1986-12-17 1988-09-28 Rixhter Gedeon Vegyeszeti Gyar Cleaned active substances of biological origin hindering the nutrition selectively, their antibodies and immune complexes of these active substances and the proper antibodies
JP6930101B2 (ja) * 2016-12-12 2021-09-01 オムロン株式会社 音響センサ及び静電容量型トランスデューサ

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
HU178703B (en) * 1978-08-29 1982-06-28 Richter Gedeon Vegyeszet Process for separating appetite-controlling fraction from human or animal sera,of activity specifically on the nutrient center

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AT391807B (de) * 1982-05-21 1990-12-10 Solco Basel Ag Verfahren zur gewinnung zellatmungsfoerdernder wirkstoffe aus kaelberblut

Also Published As

Publication number Publication date
JPH0427998B2 (fr) 1992-05-13
AU551250B2 (en) 1986-04-24
DK429082A (da) 1983-03-29
EP0075925A2 (fr) 1983-04-06
DE3280277D1 (de) 1991-02-07
ATE59556T1 (de) 1991-01-15
AU8877482A (en) 1983-05-12
HU183590B (en) 1984-05-28
US4430264A (en) 1984-02-07
JPS58135816A (ja) 1983-08-12
EP0075925A3 (en) 1986-01-02

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